Folate is a key regulator of one-carbon metabolism (OCM), a biochemical pathway that provides methyl groups for numerous reactions including hundreds of essential methyltransferase enzymes. Food fortification with folic acid (FA), a synthetic inactive form of folate more stable than the natural form in food (5-methyltetrahydrofolate (5mTHF)), is mandated by law in 87 countries, including the US. People vary in their ability to metabolize FA to 5mTHF, resulting in unmetabolized FA (UMFA), which is present in serum of >95% of the US population. While adequate folate intake is essential for human health, the widespread presence of UMFA in serum has raised questions regarding potential unanticipated adverse effects. Expert panels systematically reviewing the safety of high FA intake concluded there is strong evidence on the benefits of FA, e.g. for neural tube defect prevention, but uncertainty for non-NTD outcomes. Many feel that there is a critical need to identify alterations in metabolites and metabolic pathways associated with high FA intake. The metabolome offers a robust approach to do so as it integrates meaningful changes in a broad spectrum of key regulatory processes. The goal of this proposal is to leverage a wealth of data and banked samples from a recently completed randomized, double-blind, placebo-controlled trial (RCT) of FA supplementation (FACT, n=610) in Bangladesh. Unlike the US, Bangladesh is a FA-nave population, as foods are not fortified with FA. We propose to incorporate new studies that will use novel high-resolution metabolomics (HRM). Untargeted metabolomics is the quantitative measurement of small-molecule metabolites that captures an unbiased snap-shot of the activity of all metabolically active organ systems involved in the development of almost all metabolic disorders. The FACT study design includes supplementation with FA (400 or 800 g/d x 12 or 24 weeks), and a ?wash-out? period following FA cessation. This study design permits us to identify and validate novel metabolites and pathways altered by FA supplementation (Aim 1) and UMFA (Aim 2) and to determine the stability or reversibility of those effects over time. In collaboration with Dr. Walker, leader of the high-resolution metabolomics facility at Mt. Sinai, we will combine FACT?s rigorous RCT approach ? the gold standard design to determine causality ? with HRM that interrogates greater than 80% of metabolic pathways. We will test the hypotheses that FA supplementation and/or UMFA influence unanticipated downstream metabolites and pathways, and identify those that may be linked to health outcomes. In Aim 3, in collaboration with geneticist, Dr. Pierce, we will evaluate how gene variants influence the effects of FA supplementation and UMFA on metabolomic outcomes. In Aim 4, with Dr. Kioumourtzoglou, we will use novel pattern recognition and hierarchical approaches to identify specific metabolic patterns that are impacted by FA supplementation and/or UMFA. The findings of this study may inform policy decisions regarding FA fortification programs and the forms and doses of folate sold in over-the-counter supplements and used in popular beverage products.